Lecture 2: Bacterial Polymerization

Reading assignments in Text: Lengeler et al. 1999Text: pages 343-352 DNA replicationText: pages 362-368, 441 RNA transcriptionText: pages 369-376 Translation

Lecture 1 Reading assignments in Text: Lengeler et al. 1999Text: pages 110-113 Metabolic overviewText: pages 568-569, 573-574 Pili (Fimbriae) and flagellaText: pages 825-829 Surface virulence factors

Recap and prospectus

AssemblyFuelling Biosyn. Polymer.

Lecture 1

Pili

Flagella

Lecture 2Lectures 3,4

Pili = “extra-cellular microtubules”

pap system illustrated: Protein chaperones (PapD)

Ordered substrate productionReaction

B CASubstrate

Y. pestis illustrated:

Flagella = + PumpTurbine + Propeller

Salmonella illustrated: Check-point control (Sigma:Anti-sigma)

Metabolism =

Type III secretion

Replication

DNA, RNA, Protein polymerization

Biological polymerization = Assembly (+catalysis)

DNA RNA Protein Function

Transcription Translation

? What makes a good drug ?Antibiotic ?

Bacteria People

Replicating cell

DNA (chromosome) replication

originforks

Precise

Processive (clamped from origin to end)

Factory model

(proofreading, <1 error/ 4x10 bp)6

Rep- GFP

Resting cell

Replication forks

5’

3’

DnaB (helicase)

5’Leading strand

DNA Pol IIIDnaN (clamp)

RNA primer

Okazaki fragments on lagging strand

DNA Pol III

Clamp loading complex

Primase

DNA Pol IDNAGyrase

Back

DNA Gyrase

A2B2

Tyr~DNAFront

Grab Cut/ hold Ligate

-G Tension at forks

ATPOnly bacterialtopo-isomerases

A

E. coli

B Sub-units

A

Yeast Topo 2

B

Nalidixic acid (Nx)

Novobiocin (Nov)

Nx Nov

Topo-isomerases, Gyrase and Topo IV or “give me a break …”

DNA

DNAType II

Type I

E. coli (most bacteria):

Topo-isomerases: Type I

TopATopB

Type II Gyrase

predicted “swivel” from replication fork model

major replication “swivel”chromosome partition ParC, ParD

ParA, ParB

Replication fork tension

Chromosome partition/ separationGyrase Topo IV

Nx, Nov

Biologic function ?Always essential

Topo IV

cis-grabtrans-grab

Bacterial transcriptionRNA polymerases in the 3 Kingdoms

Eubacteria Archae bacteria Eukaryotes

’ A’

B’

A

B

2x

Catalytic sub-units

>10 >10

Weakhomology

Stronghomology

“Core”

“TATA box factors”+ many others

Sigma factorsDNArecognition

28 flagellin genes

32 Heat shock

54 Nitrogen assimilation

S Stationary “growth phase”

70 most genes

e.g. E. coli

Rifampicim (Rif) blocks initiation

Rif

Transcription cycle in Eubacteria

sigma

DNA

~40 bppromoter

terminator

ppp

ribosome

ppp

uuuu 3’“hair-pin”

rho -independent termination

Core polymerase

rho factortermination

eject

= RNA/DNA helicase

Rifampicim (Rif) blocks initiation

Rif

plug inRNA cleft

5’ RNA

Translation (protein synthesis)

Proteins are made on ribosomes

Programmed by mRNA

tRNA’s decode the “genetic code”

tRNA’s mediate between the RNA and Protein “worlds”

3’

=

~A ~A

~A

ATP

“charged” Amino acid with high energy bond

Anti-codon

Aminoacyl tRNA Synthetases

>20 Synthetases

Proofread Amino acids

50 S

5’

mRNA 30 S

ppp

mRNA alignment by the Shine & Dalgarno sequence

5’

mRNA30 S

?

5’ mRNA UAUCCGAUUAAGGAACGACCAUGACGCAA...

16 S RNA

3’ 16 S RNA HO-AUUCCUCCAC...

ProteinstartShine & Dalgarno

E. coliStart

codons

~90% AUG

~9% GUG ~1% UUG

Proteincoding

MethionineValine

Leucine

Startingamino acid

f Met

f Metf Met

Uniquely eubacterial

f Met

f Met

f Met

cutting

Innate immunity receptors

Phylogeny standard

Adenylation

Antibiotics target translation

Antibiotic

1 Streptomycin

2 Tetracycline

3 Chloramphenicol

4 Erythromycin

Blocks

Initiation

Initiation

Elongation

Elongation

Binds

30 S

30 S

50 S

50 S

Prevents

mRNA binding

f Met~tRNA binding

Peptide bond formation

Ribosome translocation

? Source of antibiotics: Streptomyces sps.

Resistance genes / counteractions

R-plasmids

Counteractions

acetylation

Pump

Counteraction

(and resistance genes target antibiotics)

Adenylation

Initiation of translation

Inactive Initiation FactorsIF1, 2, 3

GTP

GTP

5’

mRNA

GTP

~M

f Met~tRNA

~M

GDP + Pi

~M70 S complex

1 Streptomycin

2 Tetracycline

StrepTet

Both block assembly reactions

Translocation

Translation elongation

~

P A~ ~

P A

~

EF-TuGTP

EF-GGTP

~

AA~ tRNA binding

Peptide bond formation

-G from Pep ~ tRNA (ATP)

nn+1

N-terminus

3 Chloramphenicol4 Erythromycin

rRNA catalysis“RNA world”

Almost THE END, Translation termination

~

Stop codons UAA

UAGUGA

Release Factors( 3 RF proteins)

GTP

ATP

GTP

Peptide bonds (+ biosyn.)

Translation / assembly reactions

-G“division of labor”

Polymerization without a nucleus

DNA

RNA

protein

membrane

Who needs a nucleus ?

Bacteria >10 x higher protein synthesis rates

“prokaryotes” vs “eukaryotes”

During rapid growth ~50% mass = proteins syn. system

3 IF’s vs 10 IF’s

Smaller ribosome = 50:50 RNA:protein

Polycistronic mRNA

What makes a good Antibiotic ?

1 Distinguish2 Block

3 Cause danger

Major Assemblies

minor assemblies

General Biosyn.

specific biosyn.

Unique genes (most, e.g. biosyn., flagella)Gene families (PBP’s, Topo’s)

Target heirarchy